This invention relates to new (3R)-3-amino-4-carboxy-butyraldehyde derivatives of general formula (I), 
wherein
X represents a C1-4 alkyloxycarbonyl, an optionally substituted phenyl-(C1-2 alkyloxy)carbonyl, a C1-4 alkylcarbonyl or an optionally substituted phenylC1-3 alkyl)-carbonyl group, n represents 1 or 0,
Y represents, in the case when n=1, a tetrapeptide of general formula Y4-Y3-Y2-Y1, a tripeptide of general formula Y3-Y2-Y1 or a dipepbide of general formula Y2-Y1 or an amino acid residue of general formula Y1, or in the case when n=0, an xcex1-hydroxyacyl-tripeptide of general formula Q4-Y3-Y2-Y1, an xcex1-hydroxyacyl-dipeptide of general formula Q3-Y2-Y1 or an xcex1-hydroxyacyl-aminoacyl residue of general formula Q2-Y1,
xe2x80x83wherein
Y1-Y4 represent a residue selected from the group comprising the following L- or D-amino acids: alanine, alloisoleucine, cyclohexyl-glycine, phenylalanine, glutamine, histidine, isoleucine, leucine, lysine, methionine, pipecolic acid, proline, tyrosine and valine, and
Q2-Q4 represent an acyl group selected from the following xcex1-hydroxyacids of R or S configuration: 2cloheptyl-2-hydroxyacetic acid, 2-cyclohexyl-2-hydroxyacetic acid, 3-cyclo-hexyllactic acid, 3-phenyllactic acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxy-3-methyl-valeric acid, mandelicacid or lactic acid,
and salts thereof formed with organic or inorganic bases, and pharmaceutical compositions containing the same.
The compounds of general formula (I) of the invention have valuable therapeutic properties, particularly an inhibitory effect on the interleukin-1xcex2 converting enzyme. Accordingly, they may be applied for the treatment of various inflammatory diseases such as arthritis, colitis, hepatitis, glomerulonephritis and myocarditis, as well as septic shock.
Particularly valuable representatives of the compounds of general formula (I) of the invention are the following:
(3R)-3-(acetyl-L-tyrosyl-L-valyi-L-alanylamino)-4-carboxy-butyraldehyde, (3R)-3-(ethoxycarbonyl-L-alanyl-L-tyrosyl-L-valyl-L-alanyl-amino)-4-carboxybutyraidehyde, (SEQ ID NO:1)
(3R)-3-(methoxycarbonyl-L-alanyl-L-tyrosyl-L-valyl-L-alanyl-amino)-4-carboxybutyraldhyde, (SEQ ID NO:1)
(3R)-3-(acetyl-L-tyrosyl-L-valyl-L-histidylamino)-4-carboxy-butyraldehyde,
(3R)-3-(acetyl-L-tyrosyl-L-valyl-L-glutaminylamino)-4-carboxybutyraldehyde,
(3R)-3-(acetyl-L-tyrosyl-L-isoleucyl-L-alanylamino)-4-carboxybutyraldehyde,
(3R)-8-(acetyl-L-tyrosyl-L-alloisoleucyl-L-alaylamino)-4-carboxybutyraldehyde,
(3R)-3-(acetyl-L-tyrosyl-L-leucyl-L-alanylamino)-4-carboxy-butyraldehyde,
(3R)-3-(acetyl-L-tyrosyl-L-methionyl-L-alanylamino)-4-carboxybutyraldehyde,
(3R)-3-(acetyl-L-tyrosyl-L-cyclohexylglycyl-L-alanylamino)-4-carboxybutyraldehyde,
(3R)-3-(acetyl-L-phenylalanyt-L-valyl-L-alany lamino)-4-carboxybutyraldehyde,
(3R)-3[(2S)-(2-hydroxypropionyl)-L-tyrosyl-L-valyl-L-alanyl-amino]-4-carboxybutyraldehyde,
(3R)-3-[(2-hydrxypropionyl)-L-tyrosyl-L-valyl-L-pipecolinylamino]-4-carboxybutyraldehyde,
(3R)-3-[(2S)-(2-hydroxypropionyl)-L-tyrosyl-L-valyl-L-prolylamino]-4-carboxybutyraldehyde,
(3R)-3-[(2S)-(2-hydroxy-3-phenylpropionyl)-L-vayl-L-alanylamino]-4-carboxybutyraldehyde,
(3R)-3-[(2S)-(2-hydroxy-3cyclohexylpropionyl)-L-valyl-L-alanylamino]-4-carboxybutyraldehyde,
(3R)-3-[(2S)-(2-cyclohexyl-2-hydroxyacetyl)-L-alanylamino]-4-carboxybutyraldehyde,
(3R)-3-[(2S)-(2-cycloheptyl-2-hydroxyacetyl)-L-alanylamino]-4-carboxybutyraldehyde,
(3R)-3-[(2S)-(2-hydroxy-3-methylbutiryl)-L-alanylamino)]-4-carboxybutyraldehyde,
(3R)-3-[(2S,3S)-(2hydroxy-3-methylvaleryl)-L-alanylamino)]-4-carboxybutyraldehyde,
and their salts formed with organic or inorganic bases.
Particularly preferred compounds of general, formula (I) of the invention are (3R)-3-(acetyl-L-tyrosyl-L-valyl-L-alanyl-amino)-4-carboxybutyraldehyde and (3R)-3-(ethoxycarbonyl-L-alanyl-L-tyrosyl-L-valyl-L-alanylamino)-4-carboxy-butyraldehyde and their salts formed with organic or inorganic bases (SEQ ID NO:1)
The abbreviations of the amino acids, their substituents and peptides built up therefrom are in accordance with the prior art, e. g. J. Biol. Chem. 264, 668 (1989).
Amino Acids:
Arg=L-arginine [(2R)-2-amino-5-guanidino-pentanoic acid],
Ala=L-alanine [(2S)-2-aminopropionic acid],
Ale=L-alloisoleucine [(2S,3R)-2-amino-3-methylvaleric acid],
Asn=L-asparagine [(2S)-2-amino-3-carbamoylpropionic acid],
Asp=L-aspartic acid [(2S)-2-amino-3-arboxypropionic acid],
hAsp=L-xcex2-homoaspartic acid [(3R)-3-amino-4-carboxy-butyric acid],
Chg=L-2-cyclohexylglycine [(2S)-2-amino-2-cyclohexyl-acetic acid],
Gln=L-glutamine [(2S)-2-amino-4-carbamoylbutyric acid],
Glu L-glutamic acid [(2S)-2-amino-4-carboxybutyric acid],
Gly=glycine (2-aminoacetic acid),
His=L-histidine [(2S)-2-amino-3-(imidazolyl)propionic acid],
Ile=L-isoleucine [(2S,3S)-2-amino-3-methylvaleric acid],
Leu=L-leucine [(2S)-2-amino-4-methylvaleric acid)],
Lys=L-lysine [(2S)-2,6-diaminocaproic acid],
Met=L-methionine [(2S)-2-amino-4-methylmercapto-butyric acid],
Phe=3-phenyl-L-alanine [(2S)-2-amino-3-phenylpropionic acid],
Pip=L-pipecolic acid [(2S)-piperidine-2-carboxylic acid],
Pro=L-proline [(2S)pyrrolidine-2-carboxytic acid],
Ser=L-serine [(2S)-2-amino-3-hydroxypropionic acid],
Tyr=L-tyrosine [(2S)-2-amino-3-(4-hydroxyphenyl)-propionic acid],
Val=L-valine [(2S)-2-amino-3-methylbutyric acid].
xcex1-Hydroxyacids:
cHga=L-2-cycloheptylglycolic acid [2S)-2-cycloheptyl-2-hydroxyacetic acid],
Hma=L-hexahydromandelic acid [(2S)-2-cyclohexyl-2-hydroxyacetic acid],
Hmb=(2S)-2-hydroxy-3-methylbutyric acid,
Hmv=(2S)(3S)-2-hydroxy-3-methylvaleric acid],
Hpl=L-hexahydrophenyllactic acid [(2S)-2-hydroxy-3-cyciohexylpropionic acid],
Lac=L-lactic acid [(2S)-2-hydroxypropionic acid],
Man=L-mandelic acid [(2S)-2-phenyl-2-hydroxyacetic acid)],
Pla=L-phenyllactic acid [(2S)-2-hydroxy-3-phenylpropionic acid],
Pla(OH)=L-(4-hydroxyphenyl)lactic acid [(2S)-2-hydroxy-3-(4-hydroxyphenyl)propionic acid]
Substituens:
Ac=acetyl,
Boc=t-butoxycarbonyl,
Eoc=ethoxycarbonyl,
DCB=2,6-dichloro-benzoyloxy,
DMB=2,6-dimethyl-benzoyloxy,
Moc=methoxycarbonyl,
PhP=3-phenyl-propionyl,
Z=benzyloxycarbonyl,
Z(OH)=4-hydroxy-benzyloxycarbonyl.
The abbreviations of amino acids alone represent the respective L-amino acid. The D-amino acid is marked separately, e. g. 3-phenyl-D-alanine=D-Phe. The hyphen before and after the amino acid abbreviation designates a missing hydrogen atom from the amino group or a missing hydroxy group from the carboxy group, resp. Accordingly, Eoc-Ala-Tyr-Val-Ala-hAsp-H represents ethoxycarbonyl-L-alanyl-L-tyrosyl-L-valyl-L-alanyl-3-amino-4-carboxy-butyraldehyde.
Interleukin-1 (IL-1), a cytokine, formed e.g. in monocytes upon the action of infections, tissue injury or antigens, exerts manifold local and systemic effects [C. A. Dinarello: Blood 77, 1627 (1991)]. It is best known as an inflammation inducing cytokine but it also promotes the proliferation and differentiation of various cells while in other cells it influences the synthesis and release of enzymes, hormones, blood clotting factors and proteins of the acute phase. IL-1 is responsible for triggering inflammatory processes and maintaining inflammations followed by tissue injury [C. A. Dinarello and S. M. Wolff: N. Engl. J. Med., 328, 106 (1993)]. The direct pathologic role of IL-1 has been confirmed in rheumathoid arthritis and osteoarthritis (increased IL-1 production in the cells and high levels in the plasma and synovial fluid are indicators of these diseases). Furthermore, it is an important mediator in several other diseases such as cholangitis, encephalitis, endocarditis, pancreatitis and vasculitis. In other diseases such as disseminated intravascular coagulation, developing as a result of infections, and/or tissue injuries, it exerts its damaging action together with inflammatory cytokines (TNFxcex1). In further diseases the immunemodulator, immunadjuvant role of IL-1 becomes predominant, e. g. in the graft versus host disease, graft rejection, acute and delayed hypersensitivity and autoimmune diseases, e. g. type I diabetes mellitus and sclerosis multiplex. As an autocrine growth factor IL-1 is playing a role in certain neoplastic diseases, primarily in acute myelogenic leukaemia, IL-1 exists in two structurally different forms, IL-1xcex1 and IL-1xcex2, encoded by two different genes [C. J. March: Nature 315, 641 (1985)]. IL-1xcex1 and IL-1xcex2 are synthesized in the form of a precursor protein with 271 and 269 amino acid residues, resp., which are transformed by limited proteolysis into the mature protein with 158 and 153 members, resp. [B. S. Mosley et al.: J. Biol Chern. 262, 2941 (1987)]. Monocytes, macrophages, lymphocytes T and B, NK cells, astrocytes, fibroblasts, chondrocytes, endothelial cells, thymic epithelial cells and glyoma cells all are able to synthesize IL-1. The two forms of IL-1 induce basically similar effects on the same IL-1 receptor. The precursor and the mature form of IL-1a are both active while only the 153 membered mature form of IL-1xcex2 exerts biological activity [R. A. Black et al.: J. Biol Chem. 263, 9437 (1989)]. IL-1xcex2 is the agent produced in greater amounts, this is getting into the blood circulation, consequently the systemic effects of IL-1 may be attributed to IL-1xcex2.
During the activation of the IL-1xcex2 precursor (proIL-1xcex2) the enzyme inducing the conversion (ICE: IL-1xcex2 converting enzyme) is cleaving the bonds between Asp27 and Gly28 as well as Asp116 and Ala117. The resuit of the later cleavage is the 117-269 fragment, the mature, biologically active IL-1xcex2[P. R. Sleath et al.: J. Biol Chem. 265, 14526 (1990) and N. A. Thornberry et al.: Nature 356, 768 (1992)]. The amino acid sequence of the cleavage sites and their proximity: 
ICE is also synthesized intracellularly as a precursor peptide with 404 amino acid residues. In this molecule 4 Asp-X bonds are cleaved autocatalytically. The active enzyme is built up from the two 120-297 (p20) and 317-404 (p10) fragments which exist in the tetrameric form (p20)2/(p10)2 [K. P. Wilson et al.: Nature 370, 270 (1994)]. 
In the course of identification ICE proved to be a cysteine protease: it contained a functional cysteine moiety (Cys285) which could be easily alkylated and it was inhibited by peptide-diazomethylketone. This type of inhibitor can only react with cysteine proteases. ICE was not inhibited by the other traditional cysteine protease inhibitor, the epoxy type E64 [R. A. Black and al.: FEBS Lett 247, 389 (1989)] but was inhibited by the characteristic serine protease inhibitor 3,4-dichloro-isocoumarine [K. P. Wilson et al.: Nature 370, 270 (1994)]. The substrates are cleaved by ICE after the Asp residue which is represented in the general formula of the substrates (Pn- . . . -P2-P1↓-P1xe2x80x2- . . . -Pnxe2x80x2-) by P1 but the amino acid residue preceding itxe2x80x94P2xe2x80x94may be quite different (Ala, His, Gln, Lys, Asp). This role of P1 can be observed in serine proteases while the substrate specificity of cysteine proteases is more dependent on the type of P2 (L. Polgar Mechanism of Protease Action, CRC Press, Boca Raton, 1989, Chapter 4). All these findings suggest that ICE is a specific cysteine protease with properties different from those of known cysteine proteases [M. A. Ator and R. E. Dolle: Current Pharmaceutical Design 1, 191 (1995)].
Attempts to inhibit IL-1xcex2 are justified by its pathological role. Its function may be blocked by inhibiting its biosyntheses, release or receptorinding. From the manifold strategies the search for receptor antagonists has found greatest attention. Unfortunately, the low molecular weight agents most useful in therapy proved to be poor antagonists. The glucocorticoids, the best inhibitors of biosynthesis, had different main activity and, in addition, had severe side effects, too. The strategies for inhibiting IL-1 became broader with the discovery of ICE, the proIL-1xcex2 converting enzyme. By recognising the structure and function of ICE, low molecular weight inhibitors may be developed, which inhibit the autocatalysis of the enzyme, the development of active ICE and/or its proteolytic action an proIL-1xcex2 whereby no active IL-1xcex2 is released, consequently no IL1xcex2 appears in the blood circulation.
It is known that both cysteine and serine proteases may be inhibited by peptide aldehydes with an amino acid sequence which is similar to the sequence prior to the peptide bond (↓) cleaved in the substrates: Pn- . . . -P2-P1 [J. C. Powers and J. W. Harper: in Protease Inhibitors (A. J. Barrett and G. Salvesen, eds.), Elsevier, Amsterdam, 1986, Chapter 3; D. H. Rich: idem, Chapter 4]. The peptide P1 and serine protease S1 subunits, and the peptide P2 and cysteine protease S2 subunits are participating in the recognition of the respective sites. First these are coupled to one another, this is rapidly followed by the interaction of further Pn/Sn subunits, then the addition between the xcex1xe2x80x94CHO group of the P1 amino acid and the active OH group of the serine or the SH group of the cysteine in the proteases. A hemiacetal and a thiohemiacetal are formed, resp., which are non-productive analogues of the tetrahedral intermediate formed with the COxe2x80x94NH group of the substrate to be cleaved. The reaction is reversible, the peptide aldehydes are reversible inhibitors of the serine and cysteine proteases.
Analogue peptide diazomethyl-ketones or halomethyl- and acyloxy-methyl-ketones are suitable irreversible inhibitors of cysteine proteases. The C-terminal group of the P1 amino acid residue in these inhibitors is xcex1xe2x80x94COxe2x80x94CHN2 and xcex1xe2x80x94COxe2x80x94CH2xe2x80x94X (X=halogen or acyloxy group), resp. In the first phase of their reaction with cysteine proteases a thiohemiacetal is formedxe2x80x94similarly to the above reactionxe2x80x94, which is converted later into the more stable S-alkyl formxe2x80x94xcex1xe2x80x94COxe2x80x94CH2xe2x80x94 xe2x80x94, i e. the Pn- . . . -P2-P1 peptide moiety of the inhibitor is bound to the cysteine S atom of the protease by a methylene bridge.
In the following the proximity of the cleavage sites in proIL-1xcex2xe2x80x94the 23-32 (A) and 112-121 (B) sequencexe2x80x94is represented as a substrate fragment, wherein each 5 amino acid moiety preceding and following the cleavage site (↓) is indicated by P5-P1 and P1xe2x80x2-P5xe2x80x2, resp.:
P5 P4 P3 P2 P1P1xe2x80x2 P2xe2x80x2 P3xe2x80x2 P4xe2x80x2 P5xe2x80x2
A: Phe-Phe-Glu-Ala-Asp↓Ala-Pro-Lys-Gln-Met
B: Ala-Tyr-Val-His-Asp↓Ala-Pro-Val-Arg-Ser
The first reversible and irreversible inhibitors of ICE, the Ac-Tyr-Val-Ala-Asp-H tetrapeptide aidehyde and the analogue Ac-Tyr-Val-Ala-Asp-CHN2 diazomethylketone, resp., were described by N. A. Thomberry et al. [Nature 356, 768 (1992) and K. T. Chapman et al.: published European patent application No. 519,748]. It is apparent that in agreement with the above the sequence of the peptide derivatives is derived from the substrate fragments: P4 and P3 from B, P2 from A and P1 from both. In the aldehyde series the tripeptide (Z-Val-His-Asp-H) and pentapeptide (Eoc-Ala-Tyr-Val-His-Asp-H) corresponding to sequence B were prepared (together with the analogue Eoc-Ala-Tyr-Val-Ala-Asp-H containing Ala at position P2) [I. Faust et al.: in Peptides, Proceedings of the 13th American Peptide Symposium, 1993 (R. S. Hodges and J. A. Smith, eds.) ESCOM, Leyden, 1994, pp. 589-591]. Within the scope of irreversible inhibitors after the above diazomethylketone mostly acyloxymethylketones, e. g. Ac-Tyr-Val-Ala-AspCH2DMB and PhP-Val-Ala-AspCH2DMB [N. A. Thomberry et al.: Biochemistry 33, 3934 (1994)], and Z-Val-Ala-AspCH2DCB [C. V. C. Prasad et al.: Bioorg. Med. Chem. Lett 5, 315 (1995) and R. E. Dolle et al.: published European patent applications Nos. 623,592 and 623,606] were prepared. Furthermore, derivatives were synthesized wherein the aspartyl-methyloxyacyl or aspartyl-methyloxyhetero)aryl groups were coupled to other peptide residues or other non-peptide moieties [e. g. R. E. Dolle et al.: published international patent application WO 95 25,741 (1995) and published European patent application No. 644,198)].
The international publication WO 95/35308A describes compounds having a structure which is near to the structure of the compounds according to the invention. Although this reference claims compounds/peptides comprising xcex2-amino-aldehyde structure on the terminal C, which is similar to that of the compounds according to the invention, the examples of this reference illustrate exclusively compounds/peptides comprising xcex1-amino-aldehyde (ketone) structural units on the terminal C.
In in vitro cellular models IL-1xcex2 release is inhibited by both reversible and irreversible ICE inhibitors at values of IC50xe2x89xa610 xcexcM [D. K. Miller et al.: Ann. N. Y. Acad. Sci. 696, 133 (1994); P. R. Eiford et al.: Br. J. Pharmacol. 115, 601 (1995); K. Nxc3xa9meth et al.: Int. J. Immunopharmac. 17, 985 (1995)]. The in vivo efficacy of ICE inhibitors was also confirmed in the case of Ac-Tyr-Val-Ala-Asp-H and Z-Val-Ala-Asp-CH2DCB in the mouse, in the xe2x80x9ctissue chamberxe2x80x9d model as well as in lipopolysaccharide (LPS) induced fever or septic shock models [B. E. Miller et al.: J. Immunol: 154, 1331 (1994); D. S. Fletcher et al.: J. Interf. Cytok. Res 15, 243 (1995); P. R. Elford et al.: Br. J. Pharmacol. 115, 601 (1995)].
It is the objective of the present invention to prepare active inhibitors of interleukin-1xcex2 release.
It was unexpectedly found that the release of interleukin-1xcex2 from blood cells can be inhibited with the peptidyl derivatives of (3R)-3-amino-4-arboxybutyraldehyde of the invention, i. e. with peptide aldehydes wherein the P1 subunit is a xcex2-aminoaldehyde. The (3R)-3-amino-4-carboxybutyraldehyde is a homologue of the known L-aspartic acid xcex1-aldehyde [(2R)-2-amino-3arboxy-propionaldehyde)], i. e. L-xcex2-homo-aspartic acid xcex1-aldehyde (hAsp-H). The inhibiting effect of xcex2-aminoaldehydes, or their acyl or peptidyl derivatives, resp., on cysteine or serine proteases is not known yet in the literature. C. V. C. Prasad et al. [Bioorg. Med. Chem. Lett. 5, 315 (1995)] have proved that the xcex1xe2x80x94CO group of the Asp residue, the P1 amino acid moiety of the inhibitor, is playing a major role in enzyme inhibition. Namely, while Z-Asp-CH2DCB [i. e. Z-NHxe2x80x94CH(CH2COOH)xe2x80x94COxe2x80x94CH2DCB] has significant ICE inhibiting effect, Z-AhAsp-CH2DCB [i. e. Z-NHxe2x80x94CH(CH2COOH)xe2x80x94CH2xe2x80x94COxe2x80x94CH2DCB] containing a xcex2xe2x80x94CO group is ineffective.
This invention relates to new (3R)-3-amino-4-carboxy-butyraldehyde derivatives of general formula (I), wherein
X represents a C1-4 alkyloxycarbonyl, an optionally substituted phenyl-(C1-2xe2x80x2 alkyloxy)carbonyl, a C1-4 alkylcarbonyl or an optionally substituted phenyl-(C1-3 alkyl)-carbonyl group,
n represents 1 or 0,
Y represents, in the case when n=1, a tetrapeptide of general formula Y4-Y3-Y2-Y1, a tripeptide of general formula Y3-Y2-Y1 or a dipeptide of general formula Y2-Y1 or an amino acid residue of general formula Y1, or in the case when n=0, an xcex1-hydroxyacyl-tripeptide of general formula Q4-Y3-Y2-Y1, an xcex1-hydroxyacyl-dipeptide of general formula Q3-Y2-Y1 or an xcex1-hydroxyacyl-aminoacyl residue of general formula Q2-Y1,
xe2x80x83wherein
Y1-Y4 represent a residue selected from the group of the following L- or D-amino acids: alanine, alloisoleucine, cyclohexyl-glycine, phenylalanine, glutamine, histidine, isoleucine, leucine, lysine, methionine, pipecolic acid, proline, tyrosine and valine, and
Q2-Q4 represent an acyl group selected from the following xcex1-hydroxyacids of R or S configuration: 2-cycloheptyl-2-hydroxyacetic acid, 2-cyclohexyl-2-hydroxyacetic acid, 3-cyclohexyllactic acid, 3-phenyllactic acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxy-3-methyl-valeric acid, mandelic acid or lactic acid,
and salts thereof formed with organic or inorganic bases, and pharmaceutical compositions containing the same.
Compounds of the instant invention are conveniently prepared using the procedures described generally below and more explicitely described in the Examples hereinafter.
The compounds of general formula (I), wherein X, n and Y have the same meaning as above, are prepared by coupling a (3R)-3-amino-4-carboxybutyraldehyde derivative provided with suitable protecting groups, e. g. (3R)-3-amino-4-t-butoxycarbonyl-butyraidehyde diethyl acetal, with the (X)n-Y peptide residue, removing the protecting groups from the product and isolating the peptide derivative of general formula (I) in the form of an organic or inorganic salt.
(3R)-3-Amino-4-t-butoxycarbonyl-butyraidehyde diethyl acetal, a protected derivative of a (3R)-3-amino-4-carboxybutyraldehyde, a suitable key intermediate in the synthesis of compounds of general formula (I), is prepared by refluxing benzyloxycarbonyl-4-t-butyl-L-aspartyldiazo-methane in methanol solution in the presence of Ag2O, converting the thus-formed (3R)-3-(benzyloxycarbonylamino)-4-t-butoxybutyric acid methyl ester after saponification to the 3,5-dimethyl-pyrazolide, then reducing the latter compound by lithium-aluminium hydride, transforming the aldehyde produced with orthoformic acid ester into diethyl acetal, cleaving the benzyloxycarbonyl group by hydrogenation and isolating the obtained (3R)-3-amino-4-t-butoxycarbonyl-butyraldehyde diethyl acetal.
The peptide moiety of general formula (X)n-Y of the compounds of general formula (I) is prepared in the case of n=1 by starting at the C-terminal amino acid ester of general formula Y1 according to the usual methods used in peptide synthesis, and building up the desired tetrapeptide moiety of general formula Y4-Y3-Y2-Y1 tripeptide moiety of general formula Y3-Y2-Y1 or dipeptide moiety of general formula Y2-Y1 and coupling to these or, if desired, to the Y1 ester, the acyl group of general fonrula X, saponifying the obtained acyl-peptide ester or acyl-amino acid ester and isolating the produced acyl peptide or acyl-amino acid.
The peptide moiety of general formula (X)n-Y of the compounds of general formula (I) is prepared in the case of n=0 when xcex1-hydroxy-acylamino acid moieties are used as building blocks, e. g. the xcex1-hydroxy-acyl-tripeptide moiety of general formula Q4-Y3-Y2-Y1 xcex1-hydroxy-acyl-dipeptide moiety of general formula Q3-Y2Y1 or xcex1-hydroxy-acyl-aminoacyl moiety of general formula Q2-Y1, by acylating the tripeptide moiety of general formula Y3-Y2-Y1, the dipeptide moiety of general formula Y2-Y1 or amino acid moiety of general formula Y1 in ester form with an xcex1-hydroxy-acid of general formula Q4 or Q3 or Q2, protected at the xcex1-hydroxy group, e. g. with a tetrahydropyranyl group, saponifying the produced xcex1-hydroxy-acyl-peptide ester or xcex1-hydroxy-acyl-arnino acid ester and isolating the xcex1-hydroxy-acyl-tripeptide of general formula Q4-Y3-Y2-Y1 xcex1-hydroxy-acyl-dipeptide of general formula Q3-Y2-Y1 or xcex1-hydroxy-acylamino-acid of general formula Q2-Y1.
The compounds of general formula (I) of the invention, wherein X, n and Y have the same meaning as above, inhibit the release of IL-1xcex2 from e. g. human blood monocytes. An in vitro method was used for measuring, this effect of the compounds. Principle of the method: upon the action of a lipopolysaccharide (LPS), a constituent of the cell wall of Gram negative bacteria, serving as antigen, immunecells are activated in the blood and produce characteristic factors (e. g./cytokines and enzymes) and then release them partially in the environment. For the production of inflammatory cytokines (e. g. IL-1) appearing during cell activation monocytes represent the most important cellular elements in the blood. On the basis of investigations of DeForge [J. Immunol. 148, 2133 (1992)] and DeGroote [Cytokine. 4, 239 (1992)] the monocytes were not separated from the blood but heparinized whole human blood was used for the assay. The blood was incubated with the peptide aldehydes and the inducing LPS in a CO2 thernostate for 24 hours at 37xc2x0 C. After inducing the blood with LPS the amount of IL1xcex2 was measured in the blood plasma by EILISA (Enzyme Linked Immunosorbent Assay) method. The values measured in the treated groups (LPS plus peptide aldehyde) were related to the values measured in the control group (treated only with LPS). The inhibitory effect of peptide aldehydes on IL1xcex2 production was characterized by IC50 values. The IC50 values were calculated from the data of peptide aldehydes measured at 5 different concentrations. According to these data the IL-1xcex2 release from human whol blood was inhibited by Ac-Tyr-Val-Ala-hAsp-H, Eoc-Ala-Tyr-Val-Ala-hAspH (SEQ ID NO:1) and Lac-Tyr-Val-Ala-hAsp-h at IC50 values of 1.82xc2x10.79, 7.09xc2x10.10 and 11.0xc2x13.5, resp.
The compounds of the invention and their pharmaceutically acceptable salts are used for therapeutic purposes alone or preferably in the form of pharmaceutical formulations. The invention also refers to these formulations.
The pharmaceutical formulations comprise an effective amount of a compound of general formula (I) or a pharmaceutically acceptable salt thereof and known pharmaceutically acceptable carriers, filling materials, diluents andlor other pharmaceutical excipients.
The above carriers, diluents or filling materials can be water, alcohols, gelatine, lactose, saccharose, starch, pectin, magnesium stearate, stearic acid, talcum, various oils of animal or plant origin, furthermore glycols, e. g. propyleneglycol or polyethylene glycol. The pharmaceutical excipients can be preservatives, various natural or synthetic emulgeators, dispersing or wetting agents, colouring materials, flavouring agents, buffering materials, materials promoting disintegration and other materials improving the bioavailability of the active ingredient.
The pharmaceutical compositions of the invention can be prepared in usual formulations, such as oral compositions (administered through the mouth, such as tablets, capsules, powders, pills, dragees or granulates) as well as parenteral compositions (drugs administered by avoiding the gastrointestinal system, such as injections, infusions, suppositories, plasters or ointments).
The therapeutic dose level of the compounds of the invention depends on the individual health status and age of the patients and may vary accordingly; consequently, its level is fixed by the physician designing treatment. The daily oral or parenteral (e. g. i. v.) dose may be 0.01 to 1000 mg/kg body weight, preferably 0.25 to 20 mg/kg body weight.
This invention also relates to a method of treatment for patients (including man and/or mammalian animals) suffering from disorders or diseases which can be attributed to IL-1xcex2/ICE as previously described, and more specifically, a method of treatment involving the administration of IL-1xcex2/ICE inhibitors of formula (I) as the active constituents.
Accordingly, disease states in which the, ICE inhibitors of formula (I) may be useful as therapeutic agents include, but are not limited to, peptic shock, inflammatory conditions such as rheumathoid arthritis, colitis, hepatitis, glomerulo-nephritis, myocarditis, etc.
The following examples are illustrating but not limiting the scope of the invention. The Rf values recorded in the examples were determined by thin-layer chromatography, using silica gel as adsorbent (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt), in the following developing solvents:
1. Ethyl acetate
2. Ethyl acetate-hexane (1:2)
3. Ethyl acetate-pyridine-acetic acid-water (960:20:6:11)
4. Ethyl acetate-pyridine-acetic acid-water (480:20:6:11)
5. Ethyl acetate-pyridine-acetic acid-water (240:20:6:11)
6. Ethyl acetate-pyridine-acetic acid-water (120:20:6:11)
7. Ethyl acetate-pyridine-acetic acid-water (60:20:6:11)
8. Chloroform-acetic acid (95:5)
9. Chloroform-acetone (95:5)
The capacity factors (kxe2x80x2) specified in the examples were determined with the apparatus xe2x80x9cPharmacia LKB analytical HPLC System Twoxe2x80x9d as follows:
Column: VYDAC C-18 reversed phase: 10 xcexcm, 300 xc3x85, 4xc3x97250 mm.
Buffer A: 0.1 % trifluoroacetic acid in water.
Buffer B: 0.1 % trifluoroacetic acid in acetonitriie.
Gradients applied at 1 ml/min flow rate,
0-30 min. 0-60 % buffer B.
Detection of peptide content of the eluates was performed by UV light at 214 nm. Sample concentration was 1 mg/ml buffer A, injection volume 25 xcexcl.
The FAB mass spectra were recorded in a Finnigan MAT 8430 apparatus at a resolution of 1250. Parameters: voltage of ion accelerator 3 kV, temperature of ion source 25xc2x0 C., matrix m-nitrobenzylalcohol, FAB gas xenon and FAB accelerator voltage 9 kV.
The ESI positive ionisation measurements were performed in a VG, Quattro 4000 (Fisons Instrument) apparatus. The samples were dissolved in a mixture of acetonitrilexe2x80x94water (1:1) containing 0.1% of formic acid and were introduced with a 50 xcexcl samnple-loop into the ion source at a carrier solvent flow rate of 81-100 xcexcl/min. Parameters: voltage of ion accelerator 50 V, capillary potential 3.53 kV, temperature of ion source 100-120xc2x0 C., conus potential 35.0 V, ion energy 2.6 V, resolution (small/large masses) 14.0/14.2, data collection disk (16 point/Da).
NMR spectra were obtaine d with a Bruker AC250 spectrometer. Chemical shifts are recorded on a xcex4(xcex4TMS=0ppm) scale.
The specific rotations ([xcex1]D) were determined at 20xc2x0 C.